A commonly used approach to the management of locally advanced breast cancer involves the sequential use of chemotherapy, surgery, followed by radiation and adjuvant chemotherapy. With the advent of paclitaxel, there is increasing interest in the use of the drug in the neoadjuvant setting. Our preliminary data show that treatment of xenograft tumors in mice with paclitaxel leads to cell cycle arrest in M-phase that is accompanied by phosphorylation of Bcl-2 and other mitotic epitopes. Further, in the majority of epithelial tumor cells lines examined, the paclitaxel-induced mitotic arrest is transient and cells can exit from mitosis with a 4N DNA content. If the paclitaxel-exposed tumor cells are deficient in G1/S checkpoint response, in particular the cyclindependent kinase inhibitory activity of p2lcip1/wafl (p21), we have shown that they can inappropriately enter S-phase with a 4N DNA content. Our laboratory and others have demonstrated that these biochemical events are associated with decreased proliferation and increased apoptosis in the presence of paclitaxel. Thus, these biochemical events may serve as molecular markers of paclitaxel chemoresponsiveness in human tumors. We hypothesize that those patients with locally advanced breast cancer who show the greatest degree of M-phase arrest, Bcl-2 and Cdc25C phosphorylation, and low or absent p21 protein after paclitaxel treatment will be the ones who have the highest rate of complete pathologic response. Moreover, we postulate that we can find additional predictive markers of paclitaxel response by examining protein profiles of pre- and post-paclitaxel exposed tissue using the power of mass spectrometry. In sum, we hypothesize that patients with tumors that show the greatest degree of change in select molecular markers and overall protein expression patterns from pre- to post-paclitaxel treatment, will be those that go on to a complete pathologic response. These interrelated hypotheses will be tested through the following specific aims: Specific Aim 1: To predict the degree of tumor response from paclitaxel-mediated changes in markers of cell cycle position, proliferation, and apoptosis that result from paclitaxel administration in patients with locally advanced breast cancer. Specific Aim 2: To predict the degree of tumor response from paclitaxel-mediated changes in p21 protein level and/or phosphorylation in patients with locally advanced breast cancer. We will (a) determine if p21 levels or phosphorylation increase after paclitaxel administration in patients with locally advanced breast cancer and (b) evaluate upstream signaling pathways required for p21 increase and phosphorylation after paclitaxel administration in breast epithelial cells. Specific Aim 3: To evaluate protein expression profiles by mass spectrometry (MS) in biopsy material collected pre- and postpaclitaxel treatment. We predict that patients with pathologic complete response will demonstrate a greater change in protein expression patterns than patients without pathologic complete response. We postulate that there is a molecular profile that can be identified from biopsies collected pre- and postpaclitaxel treatment that will predict which patients will show the greatest degree of response to paclitaxel as measured by the degree of pathologic response at time of definitive surgery. Further, we will gain significant insight to alternative mechanisms of paclitaxel anti-tumor activity and resistance through our mass spectrometry-based analyses of protein profiles from pre- and post-paclitaxel biopsy material.